Evolutionary trajectory of transcription factors and selection of targets for metabolic engineering.

Transcription factors (TFs) provide potentially powerful tools for plant metabolic engineering as they often control multiple genes in a metabolic pathway. However, selecting the best TF for a particular pathway has been challenging, and the selection often relies significantly on phylogenetic relationships. Here, we offer examples where evolutionary relationships have facilitated the selection of the suitable TFs, alongside situations where such relationships are misleading from the perspective of metabolic engineering. We argue that the evolutionary trajectory of a particular TF might be a better indicator than protein sequence homology alone in helping decide the best targets for plant metabolic engineering efforts. This article is part of the theme issue 'The evolution of plant metabolism'.

Surgical treatment of refractory low back pain using implanted BurstDR spinal cord stimulation (SCS) in a cohort of patients without options for corrective surgery: Findings and results from the DISTINCT study, a prospective randomized multi-center-controlled trial.

Background: Low back pain (LBP) is a highly prevalent, disabling condition affecting millions of people. Patients with an identifiable anatomic pain generator and resulting neuropathic lower extremity symptoms often undergo spine surgery, but many patients lack identifiable and/or surgically corrective pathology. Nonoperative treatment options often fail to provide sustained relief. Spinal cord stimulation (SCS) is sometimes used to treat these patients, but the lack of level 1 evidence limits its widespread use and insurance coverage. The DISTINCT RCT study evaluates the efficacy of passive recharge burst SCS compared to conventional medical treatment (CMM) in alleviating chronic, refractory axial low back pain. Methods: This prospective, multicenter, randomized, study with an optional 6-month crossover involved patients who were not candidates for lumbar spine surgery. The primary and secondary endpoints evaluated improvements in low back pain intensity (NRS), back pain-related disability (ODI), pain catastrophizing (PCS), and healthcare utilization. Patients were randomized to SCS therapy or CMM at 30 US study sites. Results: The SCS arm reported an 85.3% NRS responder rate (≥ 50% reduction) compared to 6.2% (5/81) in the CMM arm. After the 6M primary endpoint, SCS patients elected to remain on assigned therapy and 66.2% (49/74) of CMM patients chose to trial SCS (crossover). At the 12M follow-up, SCS and crossover patients reported 78.6% and 71.4% NRS responder rates. Secondary outcomes indicated significant improvements in ODI, PCS, and reduced healthcare utilization. Six serious adverse events were reported and resolved without sequelae. Conclusion: DISTINCT chronic low back pain patients with no indication for corrective surgery experienced a significant and sustained response to burst SCS therapy for up to 12 months. CMM patients who crossed over to the SCS arm reported profound improvements after 6 months. This data advocates for a timely consideration of SCS therapy in patients unresponsive to conservative therapy.

Genome and life-history evolution link bird diversification to the end-Cretaceous mass extinction.

Complex patterns of genome evolution associated with the end-Cretaceous [Cretaceous-Paleogene (K-Pg)] mass extinction limit our understanding of the early evolutionary history of modern birds. Here, we analyzed patterns of avian molecular evolution and identified distinct macroevolutionary regimes across exons, introns, untranslated regions, and mitochondrial genomes. Bird clades originating near the K-Pg boundary exhibited numerous shifts in the mode of molecular evolution, suggesting a burst of genomic heterogeneity at this point in Earth's history. These inferred shifts in substitution patterns were closely related to evolutionary shifts in developmental mode, adult body mass, and patterns of metabolic scaling. Our results suggest that the end-Cretaceous mass extinction triggered integrated patterns of evolution across avian genomes, physiology, and life history near the dawn of the modern bird radiation.

Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

Investigating flock-associated mimicry: examining the evidence for, and drivers of, plumage mimicry in the greater and lesser necklaced laughingthrush.

Visual mimicry is less understood in birds than in other taxa. The interspecific social dominance mimicry (ISDM) hypothesis asserts that subordinate species resemble dominant ones to reduce aggression. Plumage mimicry has also been consistently noted in mixed-species flocks (MSFs), suggesting a connection to grouping behaviour, although it is unclear whether this is linked to ISDM. We studied greater necklaced laughingthrush (GNLT, Pterorhinus pectoralis) and lesser necklaced laughingthrush (LNLT, Garrulax monileger), which were recently placed in different genera. Measurements of 162 museum specimens showed LNLT converging in sympatry with GNLT in necklace colour, but diverging in necklace to body ratio, with proportionally smaller necklaces. The species were closely associated in six of seven MSF systems from Nepal to China. In a study of foraging behaviour in Nepal, aggression was rare between the species, LNLT followed GNLT and had lower foraging rates when further from GNLT. Our data suggest a link between this MSF-associated mimicry and ISDM, and that the subordinate LNLT may be the mimic and gain more from the resemblance. The species spend much time together in dense and poorly lit vegetation, where the LNLTs resemblance to GNLTs potentially allows them to forage closer to GNLTs than would be otherwise possible.

Healthcare Utilization (HCU) Reduction with High-Frequency (10 kHz) Spinal Cord Stimulation (SCS) Therapy.

Spinal cord stimulation (SCS) is a well-established treatment for patients with chronic pain. With increasing healthcare costs, it is important to determine the benefits of SCS in healthcare utilization (HCU). This retrospective, single-center observational study involved 160 subjects who underwent implantation of a high-frequency (10 kHz) SCS device. We focused on assessing trends in HCU by measuring opioid consumption in morphine milligram equivalents (MME), as well as monitoring emergency department (ED) and office visits for interventional pain procedures during the 12-month period preceding and following the SCS implant. Our results revealed a statistically significant reduction in HCU in all domains assessed. The mean MME was 51.05 and 26.52 pre- and post-implant, respectively. There was a 24.53 MME overall decrease and a mean of 78.2% statistically significant dose reduction (p < 0.0001). Of these, 91.5% reached a minimally clinically important difference (MCID) in opioid reduction. Similarly, we found a statistically significant (p < 0.01) decrease in ED visits, with a mean of 0.12 pre- and 0.03 post-implant, and a decrease in office visits for interventional pain procedures from a 1.39 pre- to 0.28 post-10 kHz SCS implant, representing a 1.11 statistically significant (p < 0.0001) mean reduction. Our study reports the largest cohort of real-world data published to date analyzing HCU trends with 10 kHz SCS for multiple pain etiologies. Furthermore, this is the first and only study evaluating HCU trends with 10 kHz SCS by assessing opioid use, ED visits, and outpatient visits for interventional pain procedures collectively. Preceding studies have individually investigated these outcomes, consistently yielding positive results comparable to our findings.

A region of suppressed recombination misleads neoavian phylogenomics.

Genomes are typically mosaics of regions with different evolutionary histories. When speciation events are closely spaced in time, recombination makes the regions sharing the same history small, and the evolutionary history changes rapidly as we move along the genome. When examining rapid radiations such as the early diversification of Neoaves 66 Mya, typically no consistent history is observed across segments exceeding kilobases of the genome. Here, we report an exception. We found that a 21-Mb region in avian genomes, mapped to chicken chromosome 4, shows an extremely strong and discordance-free signal for a history different from that of the inferred species tree. Such a strong discordance-free signal, indicative of suppressed recombination across many millions of base pairs, is not observed elsewhere in the genome for any deep avian relationships. Although long regions with suppressed recombination have been documented in recently diverged species, our results pertain to relationships dating circa 65 Mya. We provide evidence that this strong signal may be due to an ancient rearrangement that blocked recombination and remained polymorphic for several million years prior to fixation. We show that the presence of this region has misled previous phylogenomic efforts with lower taxon sampling, showing the interplay between taxon and locus sampling. We predict that similar ancient rearrangements may confound phylogenetic analyses in other clades, pointing to a need for new analytical models that incorporate the possibility of such events.

High association strengths are linked to phenotypic similarity, including plumage color and patterns, of participants in mixed-species bird flocks of southwestern China.

Participants in mixed-species bird flocks (MSFs) have been shown to associate with species that are similar in body size, diet, and evolutionary history, suggesting that facilitation structures these assemblages. In addition, several studies have suggested that species in MSFs resemble each other in their plumage, but this question has not been systematically investigated for any MSF system. During the nonbreeding season of 2020 and 2021, we sampled 585 MSFs on 14 transects in 2 habitats of Tongbiguang Nature Reserve in western Yunnan Province, China. We performed social network analysis and the Multiple Regression Quadratic Assignment Procedure to evaluate the effect of 4 species traits (body size, overall plumage color, distinctive plumage patterns, and diet) and evolutionary history on species association strength at the whole-MSF and within-MSF levels. All 41 significant relationships showed that species with stronger associations were more similar in their various traits. Body size had the strongest effect on association strength, followed by phylogeny, plumage patterns, and plumage color; diet had the weakest effect. Our results are consistent with the hypotheses that the benefits of associating with phenotypically similar species outweigh the potential costs of interspecific competition, and that trait matching can occur in plumage characteristics, albeit more weakly than in other traits. Several explanations exist as to why similarities in plumage may occur in MSFs, including that they could reduce predators' ability to target phenotypically "odd" individuals. Whether trait matching in plumage occurs through assortative processes in ecological time or is influenced by co-evolution requires further study.

Bare parts in the Galliformes: the evolution of a multifunctional structure.

A morphological trait can have multiple functions shaped by varying selective forces. Bare parts in birds, such as wattles, casques and combs, are known to function in both signalling and thermoregulation. Studies have demonstrated such structures are targets of sexual selection via female choice in several species of Galliformes (junglefowl, turkeys and grouse), though other studies have shown some role in thermoregulation (guineafowl). Here, we tested fundamental hypotheses regarding the evolution and maintenance of bare parts in Galliformes. Using a phylogeny that included nearly 90% of species in the order, we evaluated the role of both sexual and natural selection in shaping the function of bare parts across different clades. We found a combination of both environmental and putative sexually selected traits strongly predicted the variation of bare parts for both males and females across Galliformes. When the analysis is restricted to the largest family, Phasianidae (pheasants, junglefowl and allies), sexually selected traits were the primary predictors of bare parts. Our results suggest that bare parts are important for both thermoregulation and sexual signalling across Galliformes but are primarily under strong sexual selection within the Phasianidae.

Variables associated with nonresponders to high-frequency (10 kHz) spinal cord stimulation.

INTRODUCTION: The use of spinal cord stimulation (SCS) therapy to treat chronic pain continues to rise. Optimal patient selection remains one of the most important factors for SCS success. However, despite increased utilization and the existence of general indications, predicting which patients will benefit from neuromodulation remains one of the main challenges for this therapy. Therefore, this study aims to identify the variables that may correlate with nonresponders to high-frequency (10 kHz) SCS to distinguish the subset of patients less likely to benefit from this intervention. MATERIALS AND METHODS: This was a retrospective single-center observational study of patients who underwent 10 kHz SCS implant. Patients were divided into nonresponders and responders groups. Demographic data and clinical outcomes were collected at baseline and statistical analysis was performed for all continuous and categorical variables between the two groups to calculate statistically significant differences. RESULTS: The study population comprised of 237 patients, of which 67.51% were responders and 32.49% were nonresponders. There was a statistically significant difference of high levels of kinesiophobia, high self-perceived disability, greater pain intensity, and clinically relevant pain catastrophizing at baseline in the nonresponders compared to the responders. A few variables deemed potentially relevant, such as age, gender, history of spinal surgery, diabetes, alcohol use, tobacco use, psychiatric illness, and opioid utilization at baseline were not statistically significant. CONCLUSION: Our study is the first in the neuromodulation literature to raise awareness to the association of high levels of kinesiophobia preoperatively in nonresponders to 10 kHz SCS therapy. We also found statistically significant differences with greater pain intensity, higher self-perceived disability, and clinically relevant pain catastrophizing at baseline in the nonresponders relative to responders. It may be appropriate to screen for these factors preoperatively to identify patients who are less likely to respond to SCS. If these modifiable risk factors are present, it might be prudent to consider a pre-rehabilitation program with pain neuroscience education to address these factors prior to SCS therapy, to enhance successful outcomes in neuromodulation.

Treatment of Refractory Low Back Pain Using Passive Recharge Burst in Patients Without Options for Corrective Surgery: Findings and Results From the DISTINCT Study, a Prospective Randomized Multicenter Controlled Trial.

OBJECTIVE: Spinal cord stimulation (SCS) is effective for relieving chronic intractable pain conditions. The Dorsal spInal cord STImulatioN vs mediCal management for the Treatment of low back pain study evaluates the effectiveness of SCS compared with conventional medical management (CMM) in the treatment of chronic low back pain in patients who had not undergone and were not candidates for lumbar spine surgery. METHODS AND MATERIALS: Patients were randomized to passive recharge burst therapy (n = 162) or CMM (n = 107). They reported severe pain and disability for more than a decade and had failed a multitude of therapies. Common diagnoses included degenerative disc disease, spondylosis, stenosis, and scoliosis-yet not to a degree amenable to surgery. The six-month primary end point compared responder rates, defined by a 50% reduction in pain. Hierarchical analyses of seven secondary end points were performed in the following order: composite responder rate (numerical rating scale [NRS] or Oswestry Disability Index [ODI]), NRS, ODI, Pain Catastrophizing Scale responder rate, Patient Global Impression of Change (PGIC) responder rate, and Patient-Reported Outcome Measure Information System-29 in pain interference and physical function. RESULTS: Intention-to-treat analysis showed a significant difference in pain responders on NRS between SCS (72.6%) and CMM (7.1%) arms (p < 0.0001). Of note, 85.2% of those who received six months of therapy responded on NRS compared with 6.2% of those with CMM (p < 0.0001). All secondary end points indicated the superiority of burst therapy over CMM. A composite measure on function or pain relief showed 91% of subjects with SCS improved, compared with 16% of subjects with CMM. A substantial improvement of 30 points was observed on ODI compared with a

Can convergence in mixed-species flocks lead to evolutionary divergence? Evidence for and methods to test this hypothesis.

One of the most fundamental goals of modern biology is to achieve a deep understanding of the origin and maintenance of biodiversity. It has been observed that in some mixed-species animal societies, there appears to be a drive towards some degree of phenotypic trait matching, such as similar coloration or patterning. Here we build on these observations and hypothesize that selection in mixed-species animal societies, such as mixed-species bird flocks, may drive diversification, potentially leading to speciation. We review evidence for possible convergent evolution and even outright mimicry in flocks from southwestern China, where we have observed several cases in which species and subspecies differ from their closest relatives in traits that match particular flock types. However, understanding whether this is phenotypic matching driven by convergence, and whether this divergence has promoted biodiversity, requires testing multiple facets of this hypothesis. We propose a series of steps that can be used to tease apart alternative hypotheses to build our understanding of the potential role of convergence in diversification in participants of mixed-species societies. Even if our social convergence/divergence hypothesis is not supported, the testing at each step should help highlight alternative processes that may affect mixed-species flocks, trait evolution and possible convergence. This article is part of the theme issue 'Mixed-species groups and aggregations: shaping ecological and behavioural patterns and processes'.

Systems biology as a framework to understand the physiological and endocrine bases of behavior and its evolution-From concepts to a case study in birds.

Organismal behavior, with its tremendous complexity and diversity, is generated by numerous physiological systems acting in coordination. Understanding how these systems evolve to support differences in behavior within and among species is a longstanding goal in biology that has captured the imagination of researchers who work on a multitude of taxa, including humans. Of particular importance are the physiological determinants of behavioral evolution, which are sometimes overlooked because we lack a robust conceptual framework to study mechanisms underlying adaptation and diversification of behavior. Here, we discuss a framework for such an analysis that applies a "systems view" to our understanding of behavioral control. This approach involves linking separate models that consider behavior and physiology as their own networks into a singular vertically integrated behavioral control system. In doing so, hormones commonly stand out as the links, or edges, among nodes within this system. To ground our discussion, we focus on studies of manakins (Pipridae), a family of Neotropical birds. These species have numerous physiological and endocrine specializations that support their elaborate reproductive displays. As a result, manakins provide a useful example to help imagine and visualize the way systems concepts can inform our appreciation of behavioral evolution. In particular, manakins help clarify how connectedness among physiological systems-which is maintained through endocrine signaling-potentiate and/or constrain the evolution of complex behavior to yield behavioral differences across taxa. Ultimately, we hope this review will continue to stimulate thought, discussion, and the emergence of research focused on integrated phenotypes in behavioral ecology and endocrinology.

The Structure of Evolutionary Model Space for Proteins across the Tree of Life.

The factors that determine the relative rates of amino acid substitution during protein evolution are complex and known to vary among taxa. We estimated relative exchangeabilities for pairs of amino acids from clades spread across the tree of life and assessed the historical signal in the distances among these clade-specific models. We separately trained these models on collections of arbitrarily selected protein alignments and on ribosomal protein alignments. In both cases, we found a clear separation between the models trained using multiple sequence alignments from bacterial clades and the models trained on archaeal and eukaryotic data. We assessed the predictive power of our novel clade-specific models of sequence evolution by asking whether fit to the models could be used to identify the source of multiple sequence alignments. Model fit was generally able to correctly classify protein alignments at the level of domain (bacterial versus archaeal), but the accuracy of classification at finer scales was much lower. The only exceptions to this were the relatively high classification accuracy for two archaeal lineages: Halobacteriaceae and Thermoprotei. Genomic GC content had a modest impact on relative exchangeabilities despite having a large impact on amino acid frequencies. Relative exchangeabilities involving aromatic residues exhibited the largest differences among models. There were a small number of exchangeabilities that exhibited large differences in comparisons among major clades and between generalized models and ribosomal protein models. Taken as a whole, these results reveal that a small number of relative exchangeabilities are responsible for much of the structure of the "model space" for protein sequence evolution. The clade-specific models we generated may be useful tools for protein phylogenetics, and the structure of evolutionary model space that they revealed has implications for phylogenomic inference across the tree of life.

Exploring Conflicts in Whole Genome Phylogenetics: A Case Study Within Manakins (Aves: Pipridae).

Some phylogenetic problems remain unresolved even when large amounts of sequence data are analyzed and methods that accommodate processes such as incomplete lineage sorting are employed. In addition to investigating biological sources of phylogenetic incongruence, it is also important to reduce noise in the phylogenomic dataset by using appropriate filtering approach that addresses gene tree estimation errors. We present the results of a case study in manakins, focusing on the very difficult clade comprising the genera Antilophia and Chiroxiphia. Previous studies suggest that Antilophia is nested within Chiroxiphia, though relationships among Antilophia+Chiroxiphia species have been highly unstable. We extracted more than 11,000 loci (ultra-conserved elements and introns) from whole genomes and conducted analyses using concatenation and multispecies coalescent methods. Topologies resulting from analyses using all loci differed depending on the data type and analytical method, with 2 clades (Antilophia+Chiroxiphia and Manacus+Pipra+Machaeopterus) in the manakin tree showing incongruent results. We hypothesized that gene trees that conflicted with a long coalescent branch (e.g., the branch uniting Antilophia+Chiroxiphia) might be enriched for cases of gene tree estimation error, so we conducted analyses that either constrained those gene trees to include monophyly of Antilophia+Chiroxiphia or excluded these loci. While constraining trees reduced some incongruence, excluding the trees led to completely congruent species trees, regardless of the data type or model of sequence evolution used. We found that a suite of gene metrics (most importantly the number of informative sites and likelihood of intralocus recombination) collectively explained the loci that resulted in non-monophyly of Antilophia+Chiroxiphia. We also found evidence for introgression that may have contributed to the discordant topologies we observe in Antilophia+Chiroxiphia and led to deviations from expectations given the multispecies coalescent model. Our study highlights the importance of identifying factors that can obscure phylogenetic signal when dealing with recalcitrant phylogenetic problems, such as gene tree estimation error, incomplete lineage sorting, and reticulation events. [Birds; c-gene; data type; gene estimation error; model fit; multispecies coalescent; phylogenomics; reticulation].

A Prospective, Randomized Single-Blind Crossover Study Comparing High-Frequency 10,000 Hz and Burst Spinal Cord Stimulation.

OBJECTIVES: Although both high-frequency and burst spinal cord stimulation (SCS) have shown improved efficacy and patient satisfaction compared with conventional tonic stimulation, there are limited data directly comparing the two. This study aimed to compare both high-frequency 10,000 Hz and burst SCS in the same patients in terms of pain relief and satisfaction in those with axial back pain with or without leg pain. MATERIALS AND METHODS: This prospective, single-blind, randomized controlled trial was conducted at an outpatient pain clinic within an academic medical center. Participants were randomly allocated to one of two groups in which they trialed either burst or high-frequency 10,000 Hz SCS over five days, followed by a 24- to 48-hour washout period with no stimulation, and the alternative therapy over the remaining four days. Visual analog scale (VAS) scores were collected immediately before and after both therapy trials. Secondary end points included percentage change in VAS score and patient preference. RESULTS: Of 25 participants, those receiving burst followed by high-frequency SCS (n = 11) had a mean VAS difference of 4.73 after the first trial period and 2.86 after the second. Of those receiving high-frequency followed by burst SCS (n = 14), mean VAS difference after the first trial period was 4.00 and 1.93 after the second trial period. Four participants were withdrawn owing to lead migration. Both therapies showed statistically significant differences in pre- minus post-VAS scores and percent relief. There were no significant differences in carryover or treatment effects between the two groups. There was a statistically significant association between trial sequence and stimulator type implanted because the first stimulator trialed was more likely to be chosen. CONCLUSIONS: There were no observed differences in VAS pain score decrease when comparing burst and high-frequency 10,000 Hz SCS programming therapies. Patient preference followed an order effect, favoring the first programming therapy in the trial sequence.

Historical specimens and the limits of subspecies phylogenomics in the New World quails (Odontophoridae).

As phylogenomics focuses on comprehensive taxon sampling at the species and population/subspecies levels, incorporating genomic data from historical specimens has become increasingly common. While historical samples can fill critical gaps in our understanding of the evolutionary history of diverse groups, they also introduce additional sources of phylogenomic uncertainty, making it difficult to discern novel evolutionary relationships from artifacts caused by sample quality issues. These problems highlight the need for improved strategies to disentangle artifactual patterns from true biological signal as historical specimens become more prevalent in phylogenomic datasets. Here, we tested the limits of historical specimen-driven phylogenomics to resolve subspecies-level relationships within a highly polytypic family, the New World quails (Odontophoridae), using thousands of ultraconserved elements (UCEs). We found that relationships at and above the species-level were well-resolved and highly supported across all analyses, with the exception of discordant relationships within the two most polytypic genera which included many historical specimens. We examined the causes of discordance and found that inferring phylogenies from subsets of taxa resolved the disagreements, suggesting that analyzing subclades can help remove artifactual causes of discordance in datasets that include historical samples. At the subspecies-level, we found well-resolved geographic structure within the two most polytypic genera, including the most polytypic species in this family, Northern Bobwhites (Colinus virginianus), demonstrating that variable sites within UCEs are capable of resolving phylogenetic structure below the species level. Our results highlight the importance of complete taxonomic sampling for resolving relationships among polytypic species, often through the inclusion of historical specimens, and we propose an integrative strategy for understanding and addressing the uncertainty that historical samples sometimes introduce to phylogenetic analyses.

Categorical edge-based analyses of phylogenomic data reveal conflicting signals for difficult relationships in the avian tree.

Phylogenetic analyses fail to yield a satisfactory resolution of some relationships in the tree of life even with genome-scale datasets, so the failure is unlikely to reflect limitations in the amount of data. Gene tree conflicts are particularly notable in studies focused on these contentious nodes, and taxon sampling, different analytical methods, and/or data type effects can further confound analyses. Although many efforts have been made to incorporate biological conflicts, few studies have curated individual genes for their efficiency in phylogenomic studies. Here, we conduct an edge-based analysis of Neoavian evolution, examining the phylogenetic efficacy of two recent phylogenomic bird datasets and three datatypes (ultraconserved elements [UCEs], introns, and coding regions). We assess the potential causes for biases in signal-resolution for three difficult nodes: the earliest divergence of Neoaves, the position of the enigmatic Hoatzin (Opisthocomus hoazin), and the position of owls (Strigiformes). We observed extensive conflict among genes for all data types and datasets even after meticulous curation. Edge-based analyses (EBA) increased congruence and provided information about the impact of data type, GC content variation (GCCV), and outlier genes on each of nodes we examined. First, outlier gene signals appeared to drive different patterns of support for the relationships among the earliest diverging Neoaves. Second, the placement of Hoatzin was highly variable, although our EBA did reveal a previously unappreciated data type effect with an impact on its position. It also revealed that the resolution with the most support here was Hoatzin + shorebirds. Finally, GCCV, rather than data type (i.e., coding vs non-coding) per se, was correlated with a signal that supports monophyly of owls + Accipitriformes (hawks, eagles, and vultures). Eliminating high GCCV loci increased the signal for owls + mousebirds. Categorical EBA was able to reveal the nature of each edge and provide a way to highlight especially problematic branches that warrant a further examination. The current study increases our understanding about the contentious parts of the avian tree, which show even greater conflicts than appreciated previously.

A novel exome probe set captures phototransduction genes across birds (Aves) enabling efficient analysis of vision evolution.

The diversity of avian visual phenotypes provides a framework for studying mechanisms of trait diversification generally, and the evolution of vertebrate vision, specifically. Previous research has focused on opsins, but to fully understand visual adaptation, we must study the complete phototransduction cascade (PTC). Here, we developed a probe set that captures exonic regions of 46 genes representing the PTC and other light responses. For a subset of species, we directly compared gene capture between our probe set and low-coverage whole genome sequencing (WGS), and we discuss considerations for choosing between these methods. Finally, we developed a unique strategy to avoid chimeric assembly by using "decoy" reference sequences. We successfully captured an average of 64% of our targeted exome in 46 species across 14 orders using the probe set and had similar recovery using the WGS data. Compared to WGS or transcriptomes, our probe set: (1) reduces sequencing requirements by efficiently capturing vision genes, (2) employs a simpler bioinformatic pipeline by limiting required assembly and negating annotation, and (3) eliminates the need for fresh tissues, enabling researchers to leverage existing museum collections. We then utilized our vision exome data to identify positively selected genes in two evolutionary scenarios-evolution of night vision in nocturnal birds and evolution of high-speed vision specific to manakins (Pipridae). We found parallel positive selection of SLC24A1 in both scenarios, implicating the alteration of rod response kinetics, which could improve color discrimination in dim light conditions and/or facilitate higher temporal resolution.